EP2393940B1 - Generation of random double-strand breaks in dna using enzymes - Google Patents

Generation of random double-strand breaks in dna using enzymes Download PDF

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Publication number
EP2393940B1
EP2393940B1 EP10704044.6A EP10704044A EP2393940B1 EP 2393940 B1 EP2393940 B1 EP 2393940B1 EP 10704044 A EP10704044 A EP 10704044A EP 2393940 B1 EP2393940 B1 EP 2393940B1
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dna
vvn
nuclease
mutant
fragments
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German (de)
English (en)
French (fr)
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EP2393940A1 (en
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Pei-Chung Hsieh
Chudi Guan
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New England Biolabs Inc
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New England Biolabs Inc
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing

Definitions

  • Breaking genomic DNA into smaller distinct sizes of fragments is an important step in many sequencing technologies.
  • Current mechanical fragmentation methods such as sonication, adaptive-focused acoustics or nebulization, generate DNA fragments without base cleavage preferences. These methods, however, have the potential to damage DNA in places other than the phosphodiester bond, and have a lower efficiency of DNA recovery compared to enzymatic methods.
  • enzymatic methods such as those that rely on restriction enzymes with specific recognition sequences produce fragments of a fixed specific size that may be large or small depending on the frequency of the occurrence of the recognition sequences.
  • WO 2005/052124 describes modulation of enzyme activity by changing reciprocal stereo-geometric positions of two catalytic centers in an enzyme.
  • WO 2005/121946 describes inferring function from shotgun sequencing data.
  • EP 1564306 describes methods for fragmenting and labelling DNA.
  • a preparation in an embodiment of the invention, includes a non-specific nuclease and a T7 Endo I mutant in a unit ratio of less than 1:200.
  • the non-specific nuclease is Vibrio vulnificus (Vvn) nuclease, which has a mutation at Q69.
  • a method for generating fragments from large DNA suitable for sequencing that includes mixing a DNA of interest with a preparation, which contains a non-specific nuclease and a T7 Endo I mutant in a unit ratio of less than 1:200.
  • the large DNA is cleaved into fragments of a size suitable for sequencing.
  • an example of the non-specific nuclease for use in the method is Vvn nuclease more particularly a Vvn nuclease mutant with increased specific activity such as a mutation at Q69.
  • Fragments of the large DNA may contain one or two blunt ends and can be further modified to ligate to adapters of the type used in sequencing DNA.
  • compositions and methods are provided that rely on a mixture of enzymes in a preparation for producing double-stranded DNA fragments of approximately uniform size where the uniform size may be predetermined according to need and generated by, for example, varying the incubation time.
  • the enzyme preparation reduces the number of non-productive nicks in dsDNA fragments compared with the dsDNA product of a non specific nuclease cleavage reaction alone by including a counter-nicking activity.
  • the counter-nicking permits the creation of overhangs of defined length which will dissociate under normal dissociation conditions and which can be repaired to blunt ends for subsequent manipulation.
  • Repair may involve chewing back a 3' overhang or synthesizing by means of a polymerase, a complementary sequence to a 5' overhang. Adaptors with blunt or single nucleotides overhang may then be ligated to the modified fragments. These fragments may then be used in various sequencing platforms.
  • Random fragmentation methods in the prior art generate overhangs but these are not discretely sized and create at least two problems.
  • dissociation of the ends is not uniform as melting temperatures depend on the length and base composition of the overhang.
  • the preparation contains a non-specific nuclease and a T7endonuclease I or mutant thereof.
  • a nuclease may be used which has a preference for cleaving AT or GC base pairs, preferably a nuclease is selected which does not have a significant bias for either GC or AT.
  • An example of a nuclease belonging to this latter category is obtained from Vibrio vulnificus (Vvn) (GI: 2625684 Wu, et al. Appl Environ Microbiol 67(1): 82-8 (2001 ).
  • extracellular nucleases that may be utilized in the preparation include Dns from Vibrio cholera ( Focareta and Manning Gene 53(1): 31-40 (1987 ); NucM from Erwinia chrysanthemi ( Moulard, et al. Mol Microbiol 8(4): 685-95 (1993 ); Endo I from E. coli ( Jekel, et al Gene 154(1): 55-59 (1995 ); Dns and DnsH from Aeromonas hydrophila ( Chang et al. Gene 122(1): 175-80 (1992 ) and Dodd, et al. FEMS Microbiol Lett 173(1): 41-6 (1999 ); Wang, et al. Nucleic Acids Res 35: 584-94 (2007 ); and Wang, et al. Nucleic Acid Res. 35: 584-594 (2007 )).
  • mutations to the nuclease can result in improved specific activity.
  • mutations of Q69 of the Vvn endonuclease results in a nuclease with enhanced specific activity.
  • the examples utilize Q69S.
  • Mutant nucleases were found to be readily produced in host cells when, for example, the gene was coupled with the gene for MBP to create a fusion protein that accumulates in the periplasmic space for improved recovery of the mutant endonuclease.
  • T7 Endo I mutant to an enzyme preparation containing a non-specific nuclease had an important beneficial effect on the generation of fragments with desired properties.
  • Nicks created by non-specific nucleases that produce fragments of different sizes under denaturing conditions effectively disappeared in the presence of a T7 Endo I mutant because of its counter-nicking activity.
  • the counter-nicking activity generated fragments with ends that could readily dissociate preferably 8 nucleotides or less from the nick site.
  • Other beneficial effects of the enzyme mixture on dsDNA included production of DNA fragments which had a predictable overhang disposition and length that were suitable for repair or removal to permit attachment of adaptors that is sometimes required for DNA sequencing platforms.
  • a plurality of nucleases are combined in a reaction mixture where at least one nuclease is of the type capable of introducing random nicks throughout the DNA on either strand and a second nuclease is capable of counter-nicking in the immediate vicinity of this first nick, but in the opposite strand of the DNA duplex, thus causing a double-stranded DNA break.
  • Plasmid DNA and different types of genomic DNA were enzymatically cleaved into DNA fragments of a size suitable for sequencing methodologies using an enzyme preparation as described above. Following the fragmentation of the DNA, DNA fragments were gel-isolated and processed for next generation sequencing.
  • the assay provided in Example 2 may be used to identify the appropriate amount of nucleases for a selected time of incubation for any particular DNA or an appropriate time of incubation for a selected ratio of nucleases.
  • the unit ratio of the two nucleases is less than 1:200, for example less than 1:100, for example, less than 1:10.
  • the range may be 1:2 to 1:200.
  • T7 Endo I or mutant thereof was defined as the amount of enzyme required to convert 90% of 2 ⁇ g of the linear-nicked 2.44 kb dsDNA into two fragments (1.37 kb and 1.07 kb) at 37°C for 1 hour.
  • Vvn nuclease and mutants thereof is defined as the amount of enzyme required to release 1 A 260 unit of acid soluble oligonucleotides in 30 min at 37°C.
  • the optimal time of incubation for the DNA fragmentation reaction may be assayed according to whether the DNA falls in the range of greater than 60% GC (high GC content), 40%-60% GC (standard GC content), or less than 40% GC (low GC content).
  • the incubation time range may be typically in the range of 10 minutes to 120 minutes, for example, 15 minutes to 60 minutes.
  • “Large” constitutes a DNA having a size which requires fragmentation for sequencing.
  • T7 Endo I mutant refers to a T7 Endo I with a mutation in the bridge region between the two catalytic domains.
  • MBP-T7 Endo I mutant acts the same as T7 Endo I.
  • MBP-Vvn nuclease acts the same as Vvn nuclease (WT or mutant).
  • Non-specific nuclease refers to any DNA nuclease that does not recognize a specific DNA sequence.
  • a DNA sequence consists of at least 2 nucleotides in a defined order. This excludes restriction endonucleases that recognize specific DNA sequences.
  • Example 1 Preparation of an enzyme mixture containing Vvn or mutant thereof
  • Vvn gene absent its signal peptide, corresponds to amino acids 19 through 231 in Figure 5 (SEQ ID NO:5). It was synthesized by PCR amplification using a pair of primers 1 (5'-AAGGTT GAATTC GCGCCACCTAGCTCCTTCTCT GCC-3') (SEQ ID NO:1) and 2 (5'-GGTAGA GGATCC TTATTGAGTTTGACAG GATTGCTG-3') (SEQ ID NO:2) and the fragment was cloned between the EcoRI and BamHI of pMALp4x vector (NEB, Ipswich, MA, #N8104).
  • the fusion protein MBP-Vvn endonuclease was expressed in the periplasmic compartment of E. coli and purified by an amylose affinity column to homogeneity.
  • the MBP-Vvn nuclease (WT) was further purified by a Heparin column (GE Healthcare, Piscataway, NJ). Protein concentration was determined by a Bradford assay (Bio-Rad Laboratories, Inc., Hercules, CA).
  • a mutant MBP-Vvn endonuclease (Q69S) was created, which had 5-10 fold greater specific activity than MBP-Vvn endonuclease (WT) ( Figures 3A - 3B ).
  • primers 3 (5'-C AAGTACGCAAAAGCCAAACTCGCGCAT CG-3') (SEQ ID NO:3) and 2 (SEQ ID NO:2) were used to amplify the C-terminal part of the Vvn gene
  • primers 1 (SEQ ID NO:1) and 4 (5'-TGCGCGAGTTTGGCTTTTGCGTA CTTGGTA-3') (SEQ ID NO:4) were used to amplify the N-terminal region of the Vvn gene, excluding the signal peptide sequence.
  • Vvn mutant endonuclease (Q69S) amino acid sequence Is shown in Figure 5 .
  • the fusion protein MBP-Vvn endonuclease (Q69S) was expressed in the periplasm of E. coli and purified by an amylose affinity column to homogeneity.
  • pNB1 In order to prepare linear dsDNA with a specific nicked site, pNB1 was utilized (2.44Kb). pNB1 is a plasmid with single sites for cleavage by Nt.BstNBI and BsaI. Cleavage with BsaI linearizes the plasmid, while Nt.BstNBI introduces a site-specific nick at its recognition site.
  • the plasmid pNB1 was digested with Nt.BstNBI and BsaI restriction enzyme at 50°C for one hour. Calf intestinal alkaline phosphatase was then added to the linear-nicked dsDNA and incubated at 37°C for one hour. This treatment prevented sealing of the Nt.BstNBI nick by E. coli ligase during the subsequent assay. Fragmented dsDNA was separated from associated enzymes using Qiagen columns (Valencia, CA).
  • T7 Endo I mutant Treatment of the fragmented pNBI DNA with the T7 Endo I mutant introduced a counter-nick into the DNA strand approximately opposite the Nt.BstNBI nick to produce two fragments (1.37 kb and 1.07 kb) (see Figure 7 ).
  • One unit of T7 Endo I mutant was defined as the amount of enzyme required to convert 90% of 2 ⁇ g of the linear-nicked 2.44 kb dsDNA into two fragments (1.37 kb and 1.07 kb) at 37°C for 1 hour with buffer containing 5 units of E.
  • Lane 5 of Figure 7 shows a reaction that satisfies this definition.
  • Vvn endonuclease mutant was added to 3 ml of a reaction mixture at 37°C containing 20 mM Tris-HCl (pH 7.5), 10 mM MgCl 2 , 0.15% Triton X-100, 50 mM NaCl, sonicated calf thymus gDNA (3 mg) and BSA (0.1 mg/ml), and incubation continued at 37°C.
  • 500 ⁇ l of reaction mixture was removed and the endonuclease activity was stopped with 500 ⁇ l of 5% TCA after 10, 20, 30, 40 and 50 minute time intervals.
  • Vvn endonuclease mutant is defined as the amount of enzyme required to release 1 A 260 unit of acid soluble oligonucleotides in 30 min at 37°C.
  • Vvn nuclease and T7 Endo I mutant were combined in a storage buffer of 10 mM Tris-HCl (pH 7.5), 50 mM NaCl, 0.1 mM EDTA, 200 ⁇ g/ml BSA and 50% glycerol at various ratios.
  • DNA fragmentation buffer contained 20 mM Tris-HCl (pH7.5), 50 mM NaCl, 10. mM MgCl 2 , 0.15% Triton X-100 and 0.1 mg/ml BSA.
  • one endonuclease was maintained in a constant amount while the other nuclease was varied in concentration under otherwise similar reaction conditions using a TCA assay.
  • the unit ratio was approximately 3:1 for MBP-T7 Endo I mutant: MBP-Vvn endonucleases (Q69S) mutant.
  • the unit ratio was decreased to 2:1, the rate of DNA degradation reduced 50% as determined by the TCA assay described above.
  • the ratio was increased to 8:1, the rate only increased 14%.
  • CB4 was shown to fragment genomic DNA from different sources and of different sizes in a time-dependent manner ( Figure 2 ).
  • CB4 is a mixture of 2 volumes of MBP-T7 Endo I mutant (PA/A) (0.26 mg/ml) and 1 volume of MBP-Vvn (WT) (0.2 mg/ml)).
  • PA/A MBP-T7 Endo I mutant
  • WT MBP-Vvn
  • CB4 also converted mixtures of small fragments with a size of 100-1500 bps into fragments of 100 to 150 bp ( Figure 2 ), a size suited to several current next generation sequencing platforms.
  • FIGS 6A and 6B show how treatment of plasmids with either MBP-Vvn nuclease (Q69S) or MBP-T7 Endo I PA/A over increasing times of incubation resulted in increased accumulation of nicked circular DNA, followed by formation of linear plasmid and degradation of this DNA into smaller fragments.
  • the desired degradation of any particular DNA is a function of the size of the starting DNA, the size of the desired fragments and the time of incubation with a selected ratio of nucleases in the mixture.

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EP10704044.6A 2009-02-03 2010-02-03 Generation of random double-strand breaks in dna using enzymes Active EP2393940B1 (en)

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US14967509P 2009-02-03 2009-02-03
US15881509P 2009-03-09 2009-03-09
US27553109P 2009-08-31 2009-08-31
PCT/US2010/023007 WO2010091060A1 (en) 2009-02-03 2010-02-03 Generation of random double strand breaks in dna using enzymes

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WO2014150938A1 (en) * 2013-03-15 2014-09-25 Dana-Farber Cancer Institute, Inc. Methods for generating nucleic acid molecule fragments having a customized size distribution
US11091758B2 (en) 2013-12-11 2021-08-17 The Regents Of The University Of California Methods for labeling DNAa fragments to reconstruct physical linkage and phase
CN104195122B (zh) * 2014-05-26 2017-01-04 杭州师范大学 Yep非特异性核酸酶、基因、载体、工程菌及应用
CN106604994B (zh) * 2014-06-23 2021-12-14 通用医疗公司 通过测序评估的DSBs的全基因组无偏鉴定(GUIDE-Seq)
US10526641B2 (en) 2014-08-01 2020-01-07 Dovetail Genomics, Llc Tagging nucleic acids for sequence assembly
WO2016134034A1 (en) 2015-02-17 2016-08-25 Dovetail Genomics Llc Nucleic acid sequence assembly
US11807896B2 (en) 2015-03-26 2023-11-07 Dovetail Genomics, Llc Physical linkage preservation in DNA storage
SG11201803289VA (en) 2015-10-19 2018-05-30 Dovetail Genomics Llc Methods for genome assembly, haplotype phasing, and target independent nucleic acid detection
DK3386550T3 (da) 2015-12-07 2021-04-26 Arc Bio Llc Fremgangsmåder til fremstilling og anvendelse af guide nukleinsyrer
KR20180116377A (ko) 2016-02-23 2018-10-24 더브테일 제노믹스 엘엘씨 게놈 어셈블리를 위한 페이징된 판독 세트의 생성 및 반수체형 페이징
US10160987B2 (en) 2016-04-07 2018-12-25 Rebecca F. McClure Composition and method for processing DNA
EP3455356B1 (en) 2016-05-13 2021-08-04 Dovetail Genomics LLC Recovering long-range linkage information from preserved samples
EP4372102A2 (en) 2016-06-06 2024-05-22 RedVault Biosciences, LP Target reporter constructs and uses thereof
US20200017862A1 (en) * 2017-03-07 2020-01-16 University Of North Carolina Charlotte Systems and methods for single-strand break signaling and repair in a cell-free system
CN107217308A (zh) * 2017-06-21 2017-09-29 北京贝瑞和康生物技术股份有限公司 一种用于检测染色体拷贝数变异的测序文库构建方法和试剂盒
CN111763664B (zh) * 2020-06-28 2021-03-26 江苏康科斯医疗科技有限公司 一种用于构建测序文库的酶反应液及其应用

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WO2005052124A2 (en) * 2003-11-21 2005-06-09 New England Biolabs, Inc. Modified dna cleaving enzymes and methods of use
US7851192B2 (en) * 2004-11-22 2010-12-14 New England Biolabs, Inc. Modified DNA cleavage enzymes and methods for use
CA2497324A1 (en) * 2004-02-17 2005-08-17 Affymetrix, Inc. Methods for fragmenting and labelling dna
US20060014179A1 (en) * 2004-06-02 2006-01-19 New England Biolabs, Inc. Inferring function from shotgun sequencing data

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CN102301009B (zh) 2015-09-30
EP2393940A1 (en) 2011-12-14
WO2010091060A1 (en) 2010-08-12
CN102301009A (zh) 2011-12-28
US8703462B2 (en) 2014-04-22

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